Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.     

A novel TLR2‐triggered signalling crosstalk synergistically intensifies TNF‐mediated IL‐6 induction

Toll‐like receptors (TLR) recognize pathogens and trigger the production of vigorous pro‐inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2‐mediated synergy, through a MyD88‐independent crosstalk, which enhances subsequent TNF‐mediated nuclear factor‐kappa B activation and interleukin‐6 induction. Membrane‐associated adaptor MAL conduces the link between TNF receptor‐associated factor 6 (TRAF6) and TNFR‐associated death domain, leading to a distinctive K63‐ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF‐mediated responses, indicating an innovative target for inflammation manipulation.     

A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71

Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3C(mut). The 3C(mut) was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3C(mut)-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detection system should greatly facilitate anti-EV71 drug discovery through a high-throughput screening.     

Inhibition of phosphorylated Ser473‐Akt from translocating into the nucleus contributes to 2‐cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis

Phosphorylated Ser473‐Akt (p‐Ser473‐Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2‐cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1‐cell embryos and found that p‐Ser473‐Akt acquired prominent nucleus localization in 2‐cell embryos physiologically. Akt specific inhibitors API‐2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2‐cell arrest at certain concentrations. 2‐cell embryos exposed to 2.0 μmol/L API‐2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p‐Ser473‐Akt in the nucleus. Simultaneously, qRT‐PCR results revealed that 2.0 μmol/L API‐2 treatment significantly downregulated the mRNA pattern of MuERV‐L and eIF‐1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p‐Ser473‐Akt during major ZGA, and Akt specific inhibitors API‐2 and MK2206 which led to 2‐cell arrest inhibited p‐Ser473‐Akt from translocating into the nucleus of 2‐cell embryos with defective ZGA as well, implying p‐Ser473‐Akt may be a potential player in the major ZGA of 2‐cell mouse embryos.     

Modulation of microglial immune responses by a novel thiourea derivative

Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of Alzheimer's disease (AD). In AD, activated microglia may facilitate the clearance of β-amyloid (Aβ), a neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of triggering neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an enhanced Aβ clearance may be beneficial for preventing the progression of the disease. Here, we show that some newly synthesized compounds may exert such a combination of functions. Using mouse primary microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial Aβ phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially available inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and N^G-methyl-l-arginine (l-NMA), show negligible effects on microglial Aβ phagocytosis. Among the thiourea derivatives, our data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting Aβ phagocytosis and in inhibiting the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low μM range). The potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The structure–activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl group largely influence the dual functions of the compounds. These findings may indicate a structural basis for the improved design of future drug therapies for AD.     

Products and bioenergy from the pyrolysis of rice straw via radio frequency plasma and its kinetics

The radio frequency plasma pyrolysis technology, which can overcome the disadvantages of common pyrolysis methods such as less gas products while significant tar formation, was used for pyrolyzing the biomass waste of rice straw. The experiments were performed at various plateau temperatures of 740, 813, 843 and 880 K with corresponding loading powers of 357, 482, 574 and 664 W, respectively. The corresponding yields of gas products (excluding nitrogen) from rice straw are 30.7, 56.6, 62.5 and 66.5 wt.% with respect to the original dried sample and the corresponding specific heating values gained from gas products are about 4548, 4284, 4469 and 4438 kcal kg^?1, respectively, for the said cases. The corresponding combustible portions remained in the solid residues are about 64.7, 35, 28.2 and 23.5 wt.% with specific heating values of 4106, 4438, 4328 and 4251 kcal kg^?1 with respective to solid residues, while that in the original dried sample is 87.2 wt.% with specific heating value of 4042 kcal kg^?1. The results indicated that the amount of combustibles converted into gas products increases with increasing plateau temperature. The kinetic model employed to describe the pyrolytic conversion of rice straw at constant temperatures agrees well with the experimental data. The best curve fittings render the frequency factor of 5759.5 s^?1, activation energy of 74.29 kJ mol^?1 and reaction order of 0.5. Data and information obtained are useful for the future design and operation of pyrolysis of rice straw via radio frequency plasma.     

大鼠睾丸中S-100蛋白生后发育变化的免疫细胞化学研究

本文应用免疫细胞方法,研究大鼠生后４天、７天、１４天、３０天、２个月和３个月睾丸中Ｓ－１００蛋白的分布和变化规律。结果表明：Ｓ－１００蛋白染色反应位于睾丸间质细胞,直到生后３０天才出现阳性细胞,数量少,着色浅,而２月龄和３月龄大鼠睾丸Ｓ－１００蛋白阳性细胞数量多,着色深。提示Ｓ－１００蛋白可能参与间质细胞的合成和分泌睾酮过程。